Antibodies Directed Against Programmed Death-1 (PD-1)

ABSTRACT

The disclosure provides antibody agents that bind to a programmed death-1 (PD-1) protein. Particular immunoglobulin heavy chain polypeptide and immunoglobulin light chain polypeptide sequences are explicitly provided. Also provided are related nucleic acids, vectors, compositions, and methods of using the anti-PD-1 antibody agent to treat a disorder or disease that is responsive to PD-1 inhibition, such as cancer or an infectious disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.16/346,485, filed on Apr. 30, 2019, which is a National Stageapplication under 35 U.S.C. § 371 of International Application No.PCT/US2017/059618, having an International Filing Date of Nov. 1, 2017,which claims the benefit of U.S. Provisional Application Ser. No.62/416,128 filed Nov. 1, 2016, and 62/427,777 filed Nov. 29, 2016. Thecontents of the prior applications are hereby incorporated by referencein their entirety.

SEQUENCE LISTING

This document contains a Sequence Listing that has been submittedelectronically as an ASCII text file named 26368-0060002 ST25.txt. TheASCII text file, created on Sep. 21, 2021, is 15 kilobytes in size. Thematerial in the ASCII text file is hereby incorporated by reference inits entirety.

BACKGROUND

Cancer is a serious public health problem, with about 595,690 people inthe United States of America expected to die of cancer in 2016 aloneaccording to the American Cancer Society, Cancer Facts & FIGS. 2016(cancer.org/acs/groups/content/@research/documents/document/acspc-047079.pdf).

BRIEF SUMMARY OF THE INVENTION

Programmed Death 1 (PD-1) (also known as Programmed Cell Death 1) is atype I transmembrane protein of 268 amino acids originally identified bysubtractive hybridization of a mouse T cell line undergoing apoptosis(Ishida et al., Embo J 11: 3887-95 (1992)). PD-1 is a member of theCD28/CTLA-4 family of T-cell regulators, and is expressed on activatedT-cells, B-cells, and myeloid lineage cells (Greenwald et al., Annu.Rev. Immunol., 23: 515-548 (2005); and Sharpe et al., Nat. Immunol., 8:239-245 (2007)).

Two ligands for PD-1 have been identified, PD ligand 1 (PD-L1) and PDligand 2 (PD-L2), both of which belong to the B7 protein superfamily(Greenwald et al, supra). PD-L1 is expressed in a variety of cell types,including cells of the lung, heart, thymus, spleen, and kidney (see,e.g., Freeman et al., J. Exp. Med., 192(7): 1027-1034 (2000); andYamazaki et al., J. Immunol., 169(10): 5538-5545 (2002)). PD-L1expression is upregulated on macrophages and dendritic cells (DCs) inresponse to lipopolysaccharide (LPS) and GM-CSF treatment, and onT-cells and B-cells upon signaling via T-cell and B-cell receptors.PD-L1 also is expressed in a variety of murine tumor cell lines (see,e.g., Iwai et al., Proc. Natl Acad. Sci. USA, 99(9): 12293-12297 (2002);and Blank et al., Cancer Res., 64(3): 1140-1145 (2004)). In contrast,PD-L2 exhibits a more restricted expression pattern and is expressedprimarily by antigen presenting cells (e.g., dendritic cells andmacrophages), and some tumor cell lines (see, e.g., Latchman et al.,Nat. Immunol., 2(3): 261-238 (2001)). High PD-L1 expression in tumors,whether on the tumor cell, stroma, or other cells within the tumormicroenvironment, correlates with poor clinical prognosis, presumably byinhibiting effector T cells and upregulating regulatory T cells (Treg)in the tumor.

PD-1 negatively regulates T-cell activation, and this inhibitoryfunction is linked to an immunoreceptor tyrosine-based switch motif(ITSM) in the cytoplasmic domain (see, e.g., Greenwald et al., supra;and Parry et al., Mol. Cell. Biol., 25: 9543-9553 (2005)). PD-1deficiency can lead to autoimmunity. For example, C57BL/6 PD-1 knockoutmice have been shown to develop a lupus-like syndrome (see, e.g.,Nishimura et al., Immunity, 11: 141-1151 (1999)). In humans, a singlenucleotide polymorphism in the PD-1 gene is associated with higherincidences of systemic lupus erythematosus, type 1 diabetes, rheumatoidarthritis, and progression of multiple sclerosis (see, e.g., Nielsen etal., Tissue Antigens, 62(6): 492-497 (2003); Bertsias et al., ArthritisRheum., 60(1): 207-218 (2009); Ni et al, Hum. Genet., 121(2): 223-232(2007); Tahoori et al., Clin. Exp. Rheumatol., 29(5): 763-767 (2011);and Kroner et al., Ann. Neurol., 58(1): 50-57 (2005)). Abnormal PD-1expression also has been implicated in T-cell dysfunctions in severalpathologies, such as tumor immune evasion and chronic viral infections(see, e.g., Barber et al., Nature, 439: 682-687 (2006); and Sharpe etal., supra).

Recent studies demonstrate that T-cell suppression induced by PD-1 alsoplays a role in the suppression of anti-tumor immunity. For example,PD-L1 is expressed on a variety of human and mouse tumors, and bindingof PD-1 to PD-L1 on tumors results in T-cell suppression and tumorimmune evasion and protection (Dong et al., Nat. Med., 8: 793-800(2002)). Expression of PD-L1 by tumor cells has been directly associatedwith their resistance to lysis by anti-tumor T-cells in vitro (Dong etal., supra; and Blank et al., Cancer Res., 64: 1140-1145 (2004)). PD-1knockout mice are resistant to tumor challenge (Iwai et al., Int.Immunol., 17: 133-144 (2005)), and T-cells from PD-1 knockout mice arehighly effective in tumor rejection when adoptively transferred totumor-bearing mice (Blank et al., supra). Blocking PD-1 inhibitorysignals using a monoclonal antibody can potentiate host anti-tumorimmunity in mice (Iwai et al., supra; and Hirano et al., Cancer Res.,65: 1089-1096 (2005)), and high levels of PD-L1 expression in tumors areassociated with poor prognosis for many human cancer types (Hamanishi etal., Proc. Natl. Acad. Sci. USA, 104: 3360-335 (2007), Brown et al, J.Immunol., 170: 1257-1266 (2003); and Flies et al., Yale Journal ofBiology and Medicine, 84(4): 409-421 (2011)).

In view of the foregoing, strategies for inhibiting PD-1 activity totreat various types of cancer and for immunopotentiation (e.g., to treatinfectious diseases) have been developed (see, e.g., Ascierto et al.,Clin. Cancer. Res., 19(5): 1009-1020 (2013)). In this respect,monoclonal antibodies targeting PD-1 have been developed for thetreatment of cancer (see, e.g., Weber, Semin. Oncol., 37(5): 430-4309(2010); and Tang et al., Current Oncology Reports, 15(2): 98-104(2013)). For example, nivolumab (also known as BMS-936558) producedcomplete or partial responses in non-small-cell lung cancer, melanoma,and renal-cell cancer in a Phase I clinical trial (see, e.g., Topalian,New England J Med., 366: 2443-2454 (2012)), and is currently in PhaseIII clinical trials. MK-3575 is a humanized monoclonal antibody directedagainst PD-1 that has shown evidence of antitumor activity in Phase Iclinical trials (see, e.g., Patnaik et al., 2012 American Society ofClinical Oncology (ASCO) Annual Meeting, Abstract #2512). In addition,recent evidence suggests that therapies which target PD-1 may enhanceimmune responses against pathogens, such as HIV (see, e.g., Porichis etal., Curr. HIV/AIDS Rep., 9(1): 81-90 (2012)). Despite these advances,however, the efficacy of these potential therapies in humans may belimited.

There is a need for additional antagonists of PD-1 (e.g., an antibody)that bind PD-1 with high affinity and effectively neutralize PD-1activity.

The present disclosure provides antibody agents and various compositionsand methods relating thereto including, for example, polypeptides,nucleic acids, cells, and various methodologies, etc.

The present invention provides novel antibodies that bind to PD-1. Insome embodiments, antibodies of the present invention bind to PD-1 withhigh affinity and effectively neutralize PD-1 activity. In someembodiments, antibody heavy chain polypeptide (SEQ ID NO:1) and lightchain polypeptide (SEQ ID NO:2) sequences are explicitly provided.

The present disclosure provides a polypeptide or an isolatedimmunoglobulin heavy chain polypeptide having an amino acid sequence asset forth in SEQ ID NO:1. The present disclosure further provides apolypeptide or an isolated immunoglobulin heavy chain polypeptide havingan amino acid sequence that shares at least about 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% overall identity with that set forth inSEQ ID NO:1. In some embodiments, sequence differences relative to thesequence set forth in SEQ ID NO:1 are not within the CDRs. In someembodiments, a polypeptide or an isolated immunoglobulin heavy chainpolypeptide includes all three CDRs of SEQ ID NO:1. In some embodiments,a polypeptide or an immunoglobulin heavy chain polypeptide includes asignal peptide. In some embodiments, a polypeptide or an immunoglobulinheavy chain polypeptide which includes a signal peptide has an aminoacid sequence as set forth in SEQ ID NO:5.

In some embodiments, a provided polypeptide or immunoglobulin heavychain polypeptide is or comprises an IgG4 polypeptide. In someembodiments, a provided polypeptide or immunoglobulin heavy chainpolypeptide comprises a human IGHG4*01 polypeptide. In some embodiments,a provided polypeptide or immunoglobulin heavy chain polypeptidecomprises one or more mutations within the IgG heavy chain region. Insome embodiments, a provided polypeptide or immunoglobulin heavy chainpolypeptide comprises an IgG4 heavy chain constant region having one ormore mutations in the heavy chain constant region. In some embodiments,a provided polypeptide or immunoglobulin heavy chain polypeptidecomprises an IgG4 heavy chain constant region having one or moremutations in hinge region. It is envisioned that in some embodiments, amutation in the IgG4 hinge region may prevent half molecule exchangewith other IgG4 molecules. In some embodiments, the one or moremutations in hinge region of IgG4 may include a serine to prolinestabilizing mutation that prevents half molecule exchange with otherIgG4 molecules. In some embodiments, the one or more mutations in hingeregion of IgG4 may include an S228P mutation. See, e.g., J. Biol. Chem.2015; 290(9):5462-5469.

The present disclosure provides a polypeptide or an isolatedimmunoglobulin light chain polypeptide having an amino acid sequence asset forth in SEQ ID NO:2. The present disclosure further provides apolypeptide or an isolated immunoglobulin light chain polypeptide havingan amino acid sequence that shares at least about 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% overall identity with that set forth inSEQ ID NO:2. In some embodiments, sequence differences relative to thesequence set forth in SEQ ID NO:2 are not within the CDRs. In someembodiments, a polypeptide or an isolated immunoglobulin light chainpolypeptide includes all three CDRs of SEQ ID NO:2. In some embodiments,a provided polypeptide or immunoglobulin light chain polypeptide is akappa light chain. In some embodiments, a provided polypeptide orimmunoglobulin light chain polypeptide comprises a human IGKC*01polypeptide. In some embodiments, a polypeptide or an immunoglobulinlight chain polypeptide includes a signal peptide. In some embodiments,a polypeptide or an immunoglobulin light chain polypeptide includes asignal peptide has an amino acid sequence as set forth in SEQ ID NO:6.

In some embodiments, the present disclosure provides an anti-PD-1antibody agent comprising at least one immunoglobulin heavy chain havingan amino acid sequence as set forth in SEQ ID NO:1 and at least oneimmunoglobulin light chain having an amino acid sequence as set forth inSEQ ID NO:2. In some embodiments an anti-PD-1 antibody agent comprisestwo immunoglobulin heavy chains, each having an amino acid sequence asset forth in SEQ ID NO:1. Alternatively or additionally, in someembodiments an anti-PD-1 antibody agent comprises two immunoglobulinlight chains, each having an amino acid sequence as set forth in SEQ IDNO:2. In some embodiments, an anti-PD-1 antibody agent has a canonicalantibody format.

In some embodiments, a provided heavy chain, light chain and/or antibodyagent is glycosylated at one or more sites. In some embodiments, aglycan is N-linked to an Fc region. In some embodiments, an antibodyagent is glycosylated at Asn297 (Kabat numbering).

In some embodiments, present disclosure provides a compositioncomprising one or more glycoforms of a heavy chain, light chain, and/orantibody agent as described herein. In some embodiments, a providedcomposition comprises plurality of glycoforms, present in specifiedabsolute and/or relative amounts. In some embodiments, the presentdisclosure provides compositions that may be substantially free of oneor more particular glycoforms of a heavy chain, light chain, and/orantibody agent as described herein.

In some embodiments, a provided heavy chain, light chain and/or antibodyagent has a structure that includes one or more disulfide bonds. In someembodiments, the one or more disulfide bonds are or include a disulfidebond at the expected position for an IgG4 immunoglobulin.

In some embodiments, an anti-PD-1 antibody agent is administered withanother antibody agent, such as one specific for lymphocyte-activationgene 3 (LAG-3) or T-cell immunoglobulin domain and mucin domain 3protein (TIM-3).

In some embodiments, an antibody agent binds to PD-1 and anotherantigen, resulting in a “dual reactive” antibody agent (e.g., abispecific antibody). For example, an antibody agent can bind to PD-1and to another negative regulator of the immune system such as, forexample, lymphocyte-activation gene 3 (LAG-3) or T-cell immunoglobulindomain and mucin domain 3 protein (TIM-3).

In addition, the present disclosure provides isolated or purifiednucleic acid sequences encoding the foregoing immunoglobulinpolypeptides, vectors comprising such nucleic acid sequences, isolatedanti-PD-1 antibody agents comprising the foregoing immunoglobulinpolypeptides, nucleic acid sequences encoding such anti-PD-1 antibodyagents, vectors comprising such nucleic acid sequences, isolated cellscomprising such vectors, compositions comprising such anti-PD-1 antibodyagents or such vectors with a pharmaceutically acceptable carrier, andmethods of treating cancer or infectious diseases in mammals byadministering effective amounts of such compositions to mammals.

BRIEF DESCRIPTION OF THE DRAWING

The Drawing included herein, which is composed of the following Figures,is for illustration purposes only not for limitation.

FIG. 1 shows a graph depicting receptor occupancy of an exemplaryanti-PD-1 antibody agent in human and cynomolgus monkey PBMCs.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present disclosure describes, at least in part, antibody agents andvarious compositions and methods relating thereto including, forexample, polypeptides, nucleic acids, cells, and various methodologies,etc. In some embodiments, antigen-binding proteins of the presentinvention bind to PD-1 with high affinity and effectively neutralizePD-1 activity. In some embodiments, immunoglobulin heavy chainpolypeptide (SEQ ID NO:1 and 5) and immunoglobulin light chainpolypeptide (SEQ ID NO:2 and 6) sequence are explicitly provided. Insome embodiments, an immunoglobulin heavy chain polypeptide and/or animmunoglobulin light chain polypeptide is isolated. The term“immunoglobulin” or “antibody,” as used herein, refers to a protein thatis found in blood or other bodily fluids of vertebrates, which is usedby the immune system to identify and neutralize foreign objects, such asbacteria and viruses. A whole immunoglobulin typically consists of fourpolypeptides: two identical copies of a heavy (H) chain polypeptide andtwo identical copies of a light (L) chain polypeptide. Each of the heavychains contains one N-terminal variable (V_(H)) region and threeC-terminal constant (C_(H)1, C_(H)2, and C_(H)3) regions, and each lightchain contains one N-terminal variable (V_(L)) region and one C-terminalconstant (C_(L)) region. Immunoglobulin light chains can be assigned toone of two distinct types, either kappa (κ) or lambda (λ), based uponthe amino acid sequences of their constant domains. In a typicalimmunoglobulin, each light chain is linked to a heavy chain bydisulphide bonds, and the two heavy chains are linked to each other bydisulphide bonds. The light chain variable region is aligned with thevariable region of the heavy chain, and the light chain constant regionis aligned with the first constant region of the heavy chain. Theremaining constant regions of the heavy chains are aligned with eachother.

The variable regions of each pair of light and heavy chains form theantigen binding site of an antibody. V_(H) and V_(L) regions have thesame general structure, with each region comprising four framework (FWor FR) regions, connected by three complementarity determining regions(CDRs). The term “framework region,” as used herein, refers to therelatively conserved amino acid sequences within the variable regionwhich are located between the hypervariable or complementary determiningregions (CDRs). In a typical immunoglobulin, there are four frameworkregions in each variable domain, which are designated FR1, FR2, FR3, andFR4. The framework regions form f3 sheets that provide the structuralframework of a variable region (see, e.g., C. A. Janeway et al. (eds.),Immunobiology, 5th Ed., Garland Publishing, New York, N.Y. (2001)).

In a typical immunoglobulin, there are three complementary determiningregions (CDRs) in each variable domain, which are designated CDR1, CDR2,and CDR3. The CDRs form the “hypervariable region” of an antibody, whichis responsible for antigen binding. The CDRs form loops connecting, andin some cases comprising part of, the β-sheet structure formed by theframework regions. While the constant regions of the light and heavychains are not directly involved in binding of the antibody to anantigen, the constant regions can influence the orientation of thevariable regions. The constant regions also exhibit various effectorfunctions, such as participation in antibody-dependentcomplement-mediated lysis or antibody-dependent cellular toxicity viainteractions with effector molecules and cells.

The disclosure provides, at least in part, antibody agents that bind toPD-1. As used herein, the term “antibody agent” refers to an agent thatspecifically binds to a particular antigen. In some embodiments, theterm encompasses any polypeptide or polypeptide complex that includesimmunoglobulin structural elements sufficient to confer specificbinding. Exemplary antibody agents include, but are not limited tomonoclonal antibodies or polyclonal antibodies. In some embodiments, anantibody agent may include one or more constant region sequences thatare characteristic of mouse, rabbit, primate, or human antibodies. Insome embodiments, an antibody agent may include one or more sequenceelements are humanized, primatized, chimeric, etc, as is known in theart. In many embodiments, the term “antibody agent” is used to refer toone or more of the art-known or developed constructs or formats forutilizing antibody structural and functional features in alternativepresentation. For example, embodiments, an antibody agent utilized inaccordance with the present invention is in a format selected from, butnot limited to, intact IgA, IgG, IgE or IgM antibodies; bi- ormulti-specific antibodies (e.g., Zybodies®, etc); antibody fragmentssuch as Fab fragments, Fab′ fragments, F(ab′)2 fragments, Fd′ fragments,Fd fragments, and isolated CDRs or sets thereof; single chain Fvs;polypeptide-Fc fusions; single domain antibodies (e.g., shark singledomain antibodies such as IgNAR or fragments thereof); cameloidantibodies; masked antibodies (e.g., Probodies®); Small ModularImmunoPharmaceuticals (“SMIPs™”); single chain or Tandem diabodies)(TandAb®; VHHs; Anticalins®; Nanobodies® minibodies; BiTE® s; ankyrinrepeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies;Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®;MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In someembodiments, an antibody may lack a covalent modification (e.g.,attachment of a glycan) that it would have if produced naturally. Insome embodiments, an antibody may contain a covalent modification (e.g.,attachment of a glycan, a payload [e.g., a detectable moiety, atherapeutic moiety, a catalytic moiety, etc], or other pendant group[e.g., poly-ethylene glycol, etc.]). In many embodiments, an antibodyagent is or comprises a polypeptide whose amino acid sequence includesone or more structural elements recognized by those skilled in the artas a complementarity determining region (CDR); in some embodiments, anantibody agent is or comprises a polypeptide whose amino acid sequenceincludes at least one CDR (e.g., at least one heavy chain CDR and/or atleast one light chain CDR) that is substantially identical to one foundin a reference antibody. In some embodiments, an included CDR issubstantially identical to a reference CDR in that it is eitheridentical in sequence or contains between 1-5 amino acid substitutionsas compared with the reference CDR. In some embodiments, an included CDRis substantially identical to a reference CDR in that it shows at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity with the reference CDR. In someembodiments, an included CDR is substantially identical to a referenceCDR in that it shows at least 96%, 96%, 97%, 98%, 99%, or 100% sequenceidentity with the reference CDR. In some embodiments, an included CDR issubstantially identical to a reference CDR in that at least one aminoacid within the included CDR is deleted, added, or substituted ascompared with the reference CDR but the included CDR has an amino acidsequence that is otherwise identical with that of the reference CDR. Insome embodiments, an included CDR is substantially identical to areference CDR in that 1-5 amino acids within the included CDR aredeleted, added, or substituted as compared with the reference CDR butthe included CDR has an amino acid sequence that is otherwise identicalto the reference CDR. In some embodiments, an included CDR issubstantially identical to a reference CDR in that at least one aminoacid within the included CDR is substituted as compared with thereference CDR but the included CDR has an amino acid sequence that isotherwise identical with that of the reference CDR. In some embodiments,an included CDR is substantially identical to a reference CDR in that1-5 amino acids within the included CDR are deleted, added, orsubstituted as compared with the reference CDR but the included CDR hasan amino acid sequence that is otherwise identical to the reference CDR.In some embodiments, an antibody agent is or comprises a polypeptidewhose amino acid sequence includes structural elements recognized bythose skilled in the art as an immunoglobulin variable domain. In someembodiments, an antibody agent is a polypeptide protein having a bindingdomain which is homologous or largely homologous to animmunoglobulin-binding domain.

In some embodiments, an anti-PD-1 antibody agent comprises animmunoglobulin heavy chain polypeptide and/or immunoglobulin light chainpolypeptide. As discussed above, programmed death 1 (PD-1) (also knownas programmed cell death 1) is a 268 amino acid type I transmembraneprotein (Ishida et al, supra). PD-1 is a member of the CD28/CTLA-4family of T-cell regulators and is expressed on activated T-cells,B-cells, and myeloid lineage cells (Greenwald et al., supra; and Sharpeet al., supra). PD-1 includes an extracellular IgV domain followed byshort extracellular stalk, a transmembrane region and an intracellulartail. The intracellular tail contains two phosphorylation sites locatedin an immunoreceptor tyrosine-based inhibitory motif and animmunoreceptor tyrosine-based switch motif, which play a role in theability of PD-1 to negatively regulate T-cell receptor signaling (see,e.g., Ishida et al., supra; and Blank et al., supra).

Certain other antibodies which bind to PD-1, and components thereof, areknown in the art (see, e.g., U.S. Pat. No. 8,168,757; Topalian et al,supra; and Patnaik et al, supra). Certain anti-PD-1 antibodies also arecommercially available from sources such as, for example, Abcam(Cambridge, Mass.).

In some embodiments, a provided heavy chain, light chain and/or antibodyagent is glycosylated and one or more sites. As used herein, “glycan” isa sugar polymer (moiety) component of a glycoprotein. The term “glycan”encompasses free glycans, including glycans that have been cleaved orotherwise released from a glycoprotein. In some embodiments, a glycan isN-linked to an Fc region. In some embodiments, an antibody agent isglycosylated at Asn297 (Kabat numbering).

In some embodiments, present disclosure provides a compositioncomprising one or more glycoforms of a heavy chain, light chain, and/orantibody agent as described herein. The term “glycoform” is used hereinto refer to a particular form of a glycoprotein. That is, when aglycoprotein includes a particular polypeptide that has the potential tobe linked to different glycans or sets of glycans, then each differentversion of the glycoprotein (i.e., where the polypeptide is linked to aparticular glycan or set of glycans) is referred to as a “glycoform.” Insome embodiments, a provided composition comprises a plurality ofglycoforms of one or more of a heavy chain, light chain, and/or antibodyagent as described herein. In some embodiments, a provided compositioncomprises a plurality of such glycoforms, present in specified absoluteand/or relative amounts. In some embodiments, the present disclosureprovides compositions that may be substantially free of one or moreparticular glycoforms of a heavy chain, light chain, and/or antibodyagent as described herein.

In some embodiments, an amount of a glycoform is expressed as a“percent.” For any given parameter, “percent” refers to the number ofmoles of a particular glycan (glycan X) relative to total moles ofglycans of a preparation. In some embodiments, “percent” refers to thenumber of moles of PNGase F-released Fc glycan X relative to total molesof PNGase F-released Fc glycans detected.

In some embodiments, a provided heavy chain, light chain and/or antibodyagent has a structure that includes one or more disulfide bonds. In someembodiments, the one or more disulfide bonds are at the expectedposition for an IgG4 immunglobulin. In some embodiments, a disulfidebond is present at one or more residues corresponding to positionsselected from residue 22, 96, 130, 143, 199, 222, 225, 257, 317, 363 and421 of SEQ ID NO: 1. In some embodiments, a disulfide bond is present atone or more residues corresponding to positions selected from residue23, 88, 134, 194 and 214 of SEQ ID NO: 2.

In some embodiments, an isolated immunoglobulin heavy chain polypeptidewhich comprises an amino acid sequence that is at least 90% identical(e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical) to SEQ ID NO: 1 or 5.

In some embodiments, an isolated immunoglobulin light chain polypeptidewhich comprises an amino acid sequence that is at least 90% identical(e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical) to SEQ ID NO: 2 or 6.

Nucleic acid or amino acid sequence “identity,” as described herein, canbe determined by comparing a nucleic acid or amino acid sequence ofinterest to a reference nucleic acid or amino acid sequence. The percentidentity is the number of nucleotides or amino acid residues that arethe same (i.e., that are identical) as between the sequence of interestand the reference sequence divided by the length of the longest sequence(i.e., the length of either the sequence of interest or the referencesequence, whichever is longer). A number of mathematical algorithms forobtaining the optimal alignment and calculating identity between two ormore sequences are known and incorporated into a number of availablesoftware programs. Examples of such programs include CLUSTAL-W,T-Coffee, and ALIGN (for alignment of nucleic acid and amino acidsequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versionsthereof) and FASTA programs (e.g., FASTA3x, FASTM, and SSEARCH) (forsequence alignment and sequence similarity searches). Sequence alignmentalgorithms also are disclosed in, for example, Altschul et al, J.Molecular Biol, 215(3): 403-410 (1990), Beigert et al, Proc. Natl. Acad.Sci. USA, 106(10): 3770-3775 (2009), Durbin et al, eds., BiologicalSequence Analysis: Probalistic Models of Proteins and Nucleic Acids,Cambridge University Press, Cambridge, UK (2009), Soding,Bioinformatics, 21(1): 951-960 (2005), Altschul et al, Nucleic AcidsRes., 25(11): 3389-3402 (1997), and Gusfield, Algorithms on Strings,Trees and Sequences, Cambridge University Press, Cambridge UK (1997)).

One or more amino acids of the aforementioned immunoglobulin heavy chainpolypeptides and/or light chain polypeptides can be replaced orsubstituted with a different amino acid. An amino acid “replacement” or“substitution” refers to the replacement of one amino acid at a givenposition or residue by another amino acid at the same position orresidue within a polypeptide sequence.

Amino acids are broadly grouped as “aromatic” or “aliphatic.” Anaromatic amino acid includes an aromatic ring. Examples of “aromatic”amino acids include histidine (H or His), phenylalanine (F or Phe),tyrosine (Y or Tyr), and tryptophan (W or Trp). Non-aromatic amino acidsare broadly grouped as “aliphatic.” Examples of “aliphatic” amino acidsinclude glycine (G or Gly), alanine (A or Ala), valine (V or Val),leucine (L or Leu), isoleucine (I or He), methionine (M or Met), serine(S or Ser), threonine (T or Thr), cysteine (C or Cys), proline (P orPro), glutamic acid (E or Glu), aspartic acid (A or Asp), asparagine (Nor Asn), glutamine (Q or Gin), lysine (K or Lys), and arginine (R orArg).

Aliphatic amino acids may be sub-divided into four sub-groups. The“large aliphatic non-polar sub-group” consists of valine, leucine, andisoleucine. The “aliphatic slightly-polar sub-group” consists ofmethionine, serine, threonine, and cysteine. The “aliphaticpolar/charged sub-group” consists of glutamic acid, aspartic acid,asparagine, glutamine, lysine, and arginine. The “small-residuesub-group” consists of glycine and alanine. The group of charged/polaramino acids may be sub-divided into three sub-groups: the“positively-charged sub-group” consisting of lysine and arginine, the“negatively-charged sub-group” consisting of glutamic acid and asparticacid, and the “polar sub-group” consisting of asparagine and glutamine.

Aromatic amino acids may be sub-divided into two sub-groups: the“nitrogen ring sub-group” consisting of histidine and tryptophan and the“phenyl sub-group” consisting of phenylalanine and tyrosine.

An amino acid replacement or substitution can be conservative,semi-conservative, or non-conservative. The phrase “conservative aminoacid substitution” or “conservative mutation” refers to the replacementof one amino acid by another amino acid with a common property. Afunctional way to define common properties between individual aminoacids is to analyze the normalized frequencies of amino acid changesbetween corresponding proteins of homologous organisms (Schulz andSchirmer, Principles of Protein Structure, Springer-Verlag, New York(1979)). According to such analyses, groups of amino acids may bedefined where amino acids within a group exchange preferentially witheach other, and therefore resemble each other most in their impact onthe overall protein structure (Schulz and Schirmer, supra).

Examples of conservative amino acid substitutions include substitutionsof amino acids within the sub-groups described above, for example,lysine for arginine and vice versa such that a positive charge may bemaintained, glutamic acid for aspartic acid and vice versa such that anegative charge may be maintained, serine for threonine such that a free—OH can be maintained, and glutamine for asparagine such that a free—NH₂ can be maintained.

“Semi-conservative mutations” include amino acid substitutions of aminoacids within the same groups listed above, but not within the samesub-group. For example, the substitution of aspartic acid forasparagine, or asparagine for lysine, involves amino acids within thesame group, but different sub-groups. “Non-conservative mutations”involve amino acid substitutions between different groups, for example,lysine for tryptophan, or phenylalanine for serine, etc.

The present disclosure provides, at least in part, an isolated anti-PD-1antibody agent comprising, consisting essentially of, or consisting ofan inventive isolated amino acid sequences described herein. As usedherein, the term “isolated” (or “purified”) refers to a nucleic acidsequence (e.g., a polynucleotide) or an amino acid sequence (e.g., apolypeptide) that is removed or separated from other components presentin its natural environment. For example, an isolated polypeptide is onethat is separated from other components of a cell in which it wasproduced (e.g., the endoplasmic reticulum or cytoplasmic proteins andRNA). An isolated polynucleotide is one that is separated from othernuclear components (e.g., histones) and/or from upstream or downstreamnucleic acid sequences. An isolated nucleic acid sequence or amino acidsequence can be at least 60% free, or at least 75% free, or at least 90%free, or at least 95% free, or at least 98% free, or at least 99% freefrom other components present in natural environment of the indicatednucleic acid sequence or amino acid sequence.

By “programmed death 1 (PD-1)-binding agent” is meant a molecule,preferably a proteinaceous molecule, that binds specifically to theprogrammed death 1 protein (PD-1). In some embodiments, a PD-1-bindingagent is an anti-PD-1 antibody agent. In some embodiments, an isolatedanti-PD-1 antibody agent comprises, consists essentially of, or consistsof an immunoglobulin heavy chain polypeptide (e.g., SEQ ID NO:1) and/oran immunoglobulin light chain polypeptide (e.g., SEQ ID NO:2). In someembodiments, an isolated anti-PD-1 antibody agent comprises, consistsessentially of, or consists of an immunoglobulin heavy chain polypeptidewhose sequence comprises SEQ ID NO:1 and an immunoglobulin light chainpolypeptide whose sequence comprises SEQ ID NO:2.

In some embodiments, a provided polypeptide or heavy chain polypeptideconsists essentially of an amino acid sequence of SEQ ID NO: 1 or SEQ IDNO: 5, and may further comprise additional components that do notmaterially affect the polypeptide, e.g., by influencing affinity of aninventive heavy chain polypeptide to PD-1. Examples of such componentsinclude, for example, protein moieties such as biotin that facilitatepurification or isolation, passenger mutations, sequences free ofproblematic sites including free cysteines, additional glycosylationsites, and high-likelihood deamidation or isomerization sites.

In some embodiments, a provided polypeptide or immunoglobulin heavychain polypeptide consists of an amino acid sequence of SEQ ID NO: 1 orSEQ ID NO: 5 and does not comprise any additional components (i.e.,components that are not endogenous to an inventive immunoglobulin heavychain polypeptide).

In some embodiments, anti-PD-1 antibody agents include variants whereone or more amino acids in the immunoglobulin heavy chain polypeptideand/or the immunoglobulin light chain polypeptide are replaced, in anycombination, with a different amino acid residue, or can be deleted orinserted, so long as the biological activity of is not materiallydiminished (e.g., enhanced or improved) as a result of the amino acidreplacements, insertions, and/or deletions. The “biological activity” ofan anti-PD-1 antibody agent refers to, for example, binding affinity forPD-1 or a particular PD-1 epitope, neutralization or inhibition of PD-1protein binding to its ligands PD-L1 and PD-L2, neutralization orinhibition of PD-1 protein activity in vivo (e.g., IC₅₀),pharmacokinetics, and cross-reactivity (e.g., with non-human homo logsor orthologs of the PD-1 protein, or with other proteins or tissues).Other biological properties or characteristics of an antigen-bindingagent recognized in the art include, for example, avidity, selectivity,solubility, folding, immunotoxicity, expression, and formulation. Theaforementioned properties or characteristics can be observed, measured,and/or assessed using standard techniques including, but not limited to,ELISA, competitive ELISA, surface plasmon resonance analysis (BIACORE™),or Kinetic Exclusion Assay (KINEXA®), in vitro or in vivo neutralizationassays, receptor-ligand binding assays, cytokine or growth factorproduction and/or secretion assays, and signal transduction andimmunohistochemistry assays.

The terms “inhibit” or “neutralize,” as used herein with respect to theactivity of an anti-PD-1 antibody agent, refer to the ability tosubstantially antagonize, prohibit, prevent, restrain, slow, disrupt,alter, eliminate, stop, or reverse the progression or severity of, forexample, the biological activity of a PD-1 protein, or a disease orcondition associated with an PD-1 protein. In some embodiment, anisolated PD-1-binding agent inhibits or neutralizes the activity of aPD-1 protein by at least about 20%, about 30%, about 40%, about 50%,about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about99%, about 100%, or a range defined by any two of the foregoing values(e.g., 20% to 100%, 40% to 100% or 60% to 95%, etc.)

In some embodiments, an anti-PD-1 antibody agent is a whole antibody ora fragment thereof (e.g., an antibody fragment). In some embodiments,the antibody or antibody fragment comprises a heavy chain constantregion that is based upon wild-type IgG1, IgG2, or IgG4 antibodies, orvariants thereof. It will be appreciated that each antibody class, orisotype, engages a distinct set of effector mechanisms for disposing ofor neutralizing antigen once recognized. As such, in some embodiments,when an anti-PD-1 antibody agent is an antibody, it can exhibit one ormore effector functions, such as participation in antibody-dependentcomplement-mediated lysis or antibody-dependent cellular toxicity viainteractions with effector molecules and cells (e.g., activation of thecomplement system).

In some embodiments, an anti-PD-1 antibody agent comprises an IgG4 heavychain constant region. In some embodiments, an anti-PD-1 antibody agentcomprises one or more mutations within the IgG heavy chain region. Insome embodiments, an anti-PD-1 antibody agent comprises an IgG4 heavychain constant region having one or more mutations in the heavy chainconstant region. In some embodiments, an anti-PD-1 antibody agentcomprises an IgG4 heavy chain constant region having one or moremutations in hinge region. It is envisioned that in some embodiments, amutation in the IgG4 hinge region may prevent half molecule exchangewith other IgG4 molecules. In some embodiments, the one or moremutations in hinge region of IgG4 may include an S228P mutation or aserine to proline stabilizing mutation that prevents half moleculeexchange with other IgG4 molecules. See, e.g., J. Biol. Chem. 2015;290(9):5462-5469.

An anti-PD-1 antibody agent also can be an antibody conjugate. In thisrespect, an anti-PD-1 antibody agent can be a conjugate of (1) ananti-PD-1 antibody and (2) a protein or non-protein moiety. For example,an anti-PD-1 antibody agent an anti-PD-1 antibody conjugated to apeptide, a fluorescent molecule, or a chemotherapeutic agent.

An anti-PD-1 antibody agent can be, or can be obtained from, a humanantibody, a non-human antibody, or a chimeric antibody. By “chimeric” ismeant an antibody or fragment thereof comprising both human andnon-human regions. In some embodiments, an anti-PD-1 antibody agent is ahumanized antibody. A “humanized” antibody is a monoclonal antibodycomprising a human antibody scaffold and at least one CDR obtained orderived from a non-human antibody. Non-human antibodies includeantibodies isolated from any non-human animal, such as, for example, arodent (e.g., a mouse or rat). A humanized antibody can comprise, one,two, or three CDRs obtained or derived from a non-human antibody. Insome embodiments, CDRH3 of an anti-PD-1 antibody agent is obtained orderived from a mouse monoclonal antibody, while the remaining variableregions and constant region of antibody agent are obtained or derivedfrom a human monoclonal antibody.

A human antibody, a non-human antibody, a chimeric antibody, or ahumanized antibody can be obtained by any means, including via in vitrosources (e.g., a hybridoma or a cell line producing an antibodyrecombinantly) and in vivo sources (e.g., rodents). Methods forgenerating antibodies are known in the art and are described in, forexample, Kohler and Milstein, Eur. J. Immunol., 5: 511-519 (1976);Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press(1988); and Janeway et al. (eds.), Immunobiology, 5th Ed., GarlandPublishing, New York, N.Y. (2001)). In certain embodiments, a humanantibody or a chimeric antibody can be generated using a transgenicanimal (e.g., a mouse) wherein one or more endogenous immunoglobulingenes are replaced with one or more human immunoglobulin genes. Examplesof transgenic mice wherein endogenous antibody genes are effectivelyreplaced with human antibody genes include, but are not limited to, theMedarex HUMAB-MOUSE™, the Kirin TC MOUSE™, and the Kyowa Kirin KM-MOUSE™(see, e.g., Lonberg, Nat. Biotechnol., 23(9): 1117-25 (2005), andLonberg, Handb. Exp. Pharmacol., 181: 69-97 (2008)). A humanizedantibody can be generated using any suitable method known in the art(see, e.g., An, Z. (ed.), Therapeutic Monoclonal Antibodies: From Benchto Clinic, John Wiley & Sons, Inc., Hoboken, N.J. (2009)), including,e.g., grafting of non-human CDRs onto a human antibody scaffold (see,e.g., Kashmiri et al, Methods, 36(1): 25-34 (2005); and Hou et al, J.Biochem., 144(1): 115-120 (2008)). In some embodiments, a humanizedantibody can be produced using the methods described in, e.g., U.S.Patent Application Publication 2011/0287485 A1.

In some embodiments, an anti-PD-1 antibody agent binds an epitope ofPD-1 which blocks the binding of PD-1 to any one or more of its putativeligands. In some embodiments, an anti-PD-1 antibody agent binds anepitope of PD-1 which blocks the binding of PD-1 to two or more of itsputative ligands. In a preferred embodiment, an anti-PD-1 antibody agentbinds an epitope of a PD-1 protein which blocks the binding of PD-1 toPD-L1 and/or PD-L2.

The disclosure also provides one or more isolated or purified nucleicacid sequences that encode an inventive immunoglobulin heavy chainpolypeptide, an inventive immunoglobulin light chain polypeptide, and/oran inventive anti-PD-1 antibody agent.

The term “nucleic acid sequence” is intended to encompass a polymer ofDNA or RNA, i.e., a polynucleotide, which can be single-stranded ordouble-stranded and which can contain non-natural or alterednucleotides. The terms “nucleic acid” and “polynucleotide” as usedherein refer to a polymeric form of nucleotides of any length, eitherribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms referto the primary structure of the molecule, and thus include double- andsingle-stranded DNA, and double- and single-stranded RNA. The termsinclude, as equivalents, analogs of either RNA or DNA made fromnucleotide analogs and modified polynucleotides such as, though notlimited to, methylated and/or capped polynucleotides. Nucleic acids aretypically linked via phosphate bonds to form nucleic acid sequences orpolynucleotides, though many other linkages are known in the art (e.g.,phosphorothioates, boranophosphates, and the like). Nucleic acidsequences encoding an inventive immunoglobulin heavy chain polypeptidesinclude, for example, SEQ ID NO: 3. Nucleic acid sequences encoding aninventive immunoglobulin light chain polypeptides include, for example,SEQ ID NO: 4.

The disclosure further provides a vector comprising one or more nucleicacid sequences encoding a PD-1-binding immunoglobulin heavy chainpolypeptide, a PD-1-binding immunoglobulin light chain polypeptide,and/or an anti-PD-1 antibody agent. The vector can be, for example, aplasmid, episome, cosmid, viral vector (e.g., retroviral or adenoviral),or phage. Suitable vectors and methods of vector preparation are wellknown in the art (see, e.g., Sambrook et al., Molecular Cloning, aLaboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (2001), and Ausubel et al, Current Protocols in MolecularBiology, Greene Publishing Associates and John Wiley & Sons, New York,N.Y. (1994)).

In addition to the nucleic acid sequence encoding an inventivepolypeptide, an inventive immunoglobulin heavy polypeptide, an inventiveimmunoglobulin light chain polypeptide, and/or an inventive—anti-PD-1antibody agent, the vector can comprise expression control sequences,such as promoters, enhancers, polyadenylation signals, transcriptionterminators, signal peptides (e.g., the osteonectin signal peptide),internal ribosome entry sites (IRES), and the like, that provide for theexpression of the coding sequence in a host cell. Exemplary expressioncontrol sequences are known in the art and described in, for example,Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185,Academic Press, San Diego, Calif. (1990).

A large number of promoters, including constitutive, inducible, andrepressible promoters, from a variety of different sources are wellknown in the art. Representative sources of promoters include forexample, virus, mammal, insect, plant, yeast, and bacteria, and suitablepromoters from these sources are readily available, or can be madesynthetically, based on sequences publicly available, for example, fromdepositories such as the ATCC as well as other commercial or individualsources. Promoters can be unidirectional (i.e., initiate transcriptionin one direction) or bi-directional (i.e., initiate transcription ineither a 3′ or 5′ direction). Non-limiting examples of promotersinclude, for example, the T7 bacterial expression system, pBAD (araA)bacterial expression system, the cytomegalovirus (CMV) promoter, theSV40 promoter, the RSV promoter. Inducible promoters include, forexample, the Tet system (U.S. Pat. Nos. 5,464,758 and 5,814,618), theEcdysone inducible system (No et al, Proc. Natl. Acad. Sci., 93:3346-3351 (1996)), the T-REX™ system (Invitrogen, Carlsbad, Calif.),LACSWITCH™ system (Stratagene, San Diego, Calif.), and the Cre-ERTtamoxifen inducible recombinase system (Indra et al, Nuc. Acid. Res.,27: 4324-4327 (1999); Nuc. Acid. Res., 28: e99 (2000); U.S. Pat. No.7,112,715; and Kramer & Fussenegger, Methods Mol. Biol, 308: 123-144(2005)).

The term “enhancer” as used herein, refers to a DNA sequence thatincreases transcription of, for example, a nucleic acid sequence towhich it is operably linked.

Enhancers can be located many kilobases away from the coding region ofthe nucleic acid sequence and can mediate the binding of regulatoryfactors, patterns of DNA methylation, or changes in DNA structure. Alarge number of enhancers from a variety of different sources are wellknown in the art and are available as or within cloned polynucleotides(from, e.g., depositories such as the ATCC as well as other commercialor individual sources). A number of polynucleotides comprising promoters(such as the commonly-used CMV promoter) also comprise enhancersequences. Enhancers can be located upstream, within, or downstream ofcoding sequences.

The vector also can comprise a “selectable marker gene.” The term“selectable marker gene,” as used herein, refers to a nucleic acidsequence that allow cells expressing the nucleic acid sequence to bespecifically selected for or against, in the presence of a correspondingselective agent. Suitable selectable marker genes are known in the artand described in, e.g., International Patent Application Publications WO1992/008796 and WO 1994/028143; Wigler et al, Proc. Natl. Acad. Sci.USA, 77: 3567-3570 (1980); O'Hare et al, Proc. Natl. Acad. Sci. USA, 78:1527-1531 (1981); Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072-2076 (1981); Colberre-Garapin et al, J. Mol. Biol., 150: 1-14(1981); Santerre et al, Gene, 30: 147-156 (1984); Kent et al, Science,237: 901-903 (1987); Wigler et al, Cell, 11: 223-232 (1977); Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA, 48: 2026-2034 (1962); Lowy et al,Cell, 22: 817-823 (1980); and U.S. Pat. Nos. 5,122,464 and 5,770,359.

In some embodiments, the vector is an “episomal expression vector” or“episome,” which is able to replicate in a host cell, and persists as anextrachromosomal segment of DNA within the host cell in the presence ofappropriate selective pressure (see, e.g., Conese et al, Gene Therapy,11: 1735-1742 (2004)). Representative commercially available episomalexpression vectors include, but are not limited to, episomal plasmidsthat utilize Epstein Barr Nuclear Antigen 1 (EBNA1) and the Epstein BarrVirus (EBV) origin of replication (oriP). The vectors pREP4, pCEP4,pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, Calif.) and pBK-CMV fromStratagene (La Jolla, Calif.) represent non-limiting examples of anepisomal vector that uses T-antigen and the SV40 origin of replicationin lieu of EBNA1 and oriP.

Other suitable vectors include integrating expression vectors, which mayrandomly integrate into the host cell's DNA, or may include arecombination site to enable the specific recombination between theexpression vector and the host cell's chromosome. Such integratingexpression vectors may utilize the endogenous expression controlsequences of the host cell's chromosomes to effect expression of thedesired protein. Examples of vectors that integrate in a site specificmanner include, for example, components of the flp-in system from LifeTechnologies (Carlsbad, Calif.) (e.g., pcDNA™5/FRT), or the cre-loxsystem, such as can be found in the pExchange-6 Core Vectors fromStratagene (La Jolla, Calif.). Examples of vectors that randomlyintegrate into host cell chromosomes include, for example, pcDNA3.1(when introduced in the absence of T-antigen) from Invitrogen (Carlsbad,Calif.), UCOE from Millipore (Billerica, Mass.), and pCI or pFN10A (ACT)FLEXI™ from Promega (Madison, Wis.).

Viral vectors also can be used. Representative commercially availableviral expression vectors include, but are not limited to, theadenovirus-based Per.C6 system available from Crucell, Inc. (Leiden, TheNetherlands), the lentiviral-based pLP1 from Invitrogen (Carlsbad,Calif.), and the retroviral vectors pFB-ERV plus pCFB-EGSH fromStratagene (La Jolla, Calif.).

Nucleic acid sequences encoding inventive amino acid sequences can beprovided to a cell on the same vector (i.e., in cis). A unidirectionalpromoter can be used to control expression of each nucleic acidsequence. In some embodiments, a combination of bidirectional andunidirectional promoters can be used to control expression of multiplenucleic acid sequences. Nucleic acid sequences encoding inventive aminoacid sequences alternatively can be provided to the population of cellson separate vectors (i.e., in trans). Each of the nucleic acid sequencesin each of the separate vectors can comprise the same or differentexpression control sequences. The separate vectors can be provided tocells simultaneously or sequentially.

The vector(s) comprising the nucleic acid(s) encoding inventive aminoacid sequences can be introduced into a host cell that is capable ofexpressing the polypeptides encoded thereby, including any suitableprokaryotic or eukaryotic cell. As such, the present disclosure providesan isolated cell comprising an inventive vector. Host cells are thosethat can be easily and reliably grown, have reasonably fast growthrates, have well characterized expression systems, and can betransformed or transfected easily and efficiently.

Examples of suitable prokaryotic cells include, but are not limited to,cells from the genera Bacillus (such as Bacillus subtilis and Bacillusbrevis), Escherichia (such as E. coli), Pseudomonas, Streptomyces,Salmonella, and Erwinia. Useful prokaryotic cells include, for example,the various strains of Escherichia coli (e.g., K12, HB101 (ATCC No.33694), DH5a, DH10, MC1061 (ATCC No. 53338), and CC102).

In some embodiments, an inventive vector is introduced into a eukaryoticcell. Suitable eukaryotic cells are known in the art and include, forexample, yeast cells, insect cells, and mammalian cells. Examples ofsuitable yeast cells include those from the genera Kluyveromyces,Pichia, Rhino-sporidium, Saccharomyces, and Schizosaccharomyces. Yeastcells include, for example, Saccharomyces cerivisae and Pichia pastoris.

Suitable insect cells are described in, for example, Kitts et al,Biotechniques, 14: 810-817 (1993); Lucklow, Curr. Opin. Biotechnol., 4:564-572 (1993); and Lucklow et al, J. Virol., 67: 4566-4579 (1993).Insect cells include, for example, Sf-9 and HI5 (Invitrogen, Carlsbad,Calif.).

In some embodiments, mammalian cells are utilized. A number of suitablemammalian host cells are known in the art, and many are available fromthe American Type Culture Collection (ATCC, Manassas, Va.). Examples ofsuitable mammalian cells include, but are not limited to, Chinesehamster ovary cells (CHO) (ATCC No. CCL61), CHO DHFR-cells (Urlaub etal, Proc. Natl. Acad. Sci. USA, 97: 4216-4220 (1980)), human embryonickidney (HEK) 293 or 293T cells (ATCC No. CRL1573), and 3T3 cells (ATCCNo. CCL92). Other suitable mammalian cell lines are the monkey COS-1(ATCC No. CRL1650) and COS-7 cell lines (ATCC No. CRL1651), as well asthe CV-1 cell line (ATCC No. CCL70).

Further exemplary mammalian host cells include primate cell lines androdent cell lines, including transformed cell lines. Normal diploidcells, cell strains derived from in vitro culture of primary tissue, aswell as primary explants, are also suitable. Other suitable mammaliancell lines include, but are not limited to, mouse neuroblastoma N2Acells, HeLa, mouse L-929 cells, and BHK or HaK hamster cell lines, allof which are available from the ATCC. Methods for selecting suitablemammalian host cells and methods for transformation, culture,amplification, screening, and purification of cells are known in theart.

In some embodiments, the mammalian cell is a human cell. For example,the mammalian cell can be a human lymphoid or lymphoid derived cellline, such as a cell line of pre-B lymphocyte origin. Examples of humanlymphoid cells lines include, without limitation, RAMOS (CRL-1596),Daudi (CCL-213), EB-3 (CCL-85), DT40 (CRL-2111), 18-81 (Jack et al,Proc. Natl. Acad. Sci. USA, 85: 1581-1585 (1988)), Raji cells (CCL-86),PER.C6 cells (Crucell Holland B. V., Leiden, The Netherlands), andderivatives thereof.

A nucleic acid sequence encoding an inventive amino acid sequence may beintroduced into a cell by “transfection,” “transformation,” or“transduction.” “Transfection,” “transformation,” or “transduction,” asused herein, refer to the introduction of one or more exogenouspolynucleotides into a host cell by using physical or chemical methods.Many transfection techniques are known in the art and include, forexample, calcium phosphate DNA co-precipitation (see, e.g., Murray E. J.(ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer andExpression Protocols, Humana Press (1991)); DEAE-dextran;electroporation; cationic liposome-mediated transfection; tungstenparticle-facilitated microparticle bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash etal, Mol. Cell Biol, 7: 2031-2034 (1987)). Phage or viral vectors can beintroduced into host cells, after growth of infectious particles insuitable packaging cells, many of which are commercially available.

The disclosure provides a composition comprising an effective amount ofan inventive immunoglobulin heavy chain polypeptide, an inventiveimmunoglobulin light chain polypeptide, an anti-PD-1 antibody agent, anucleic acid sequence encoding any of the foregoing, or a vectorcomprising the nucleic acid sequence. In some embodiments, a compositionis pharmaceutically acceptable (e.g., physiologically acceptable)composition, which comprises a carrier, preferably a pharmaceuticallyacceptable (e.g., physiologically acceptable) carrier, and an inventiveamino acid sequences, antigen-binding agent, or vector. Any suitablecarrier can be used within the context of the invention, and suchcarriers are well known in the art. The choice of carrier will bedetermined, in part, by the particular site to which the composition maybe administered and the particular method used to administer thecomposition. The composition optionally can be sterile. The compositioncan be frozen or lyophilized for storage and reconstituted in a suitablesterile carrier prior to use. The compositions can be generated inaccordance with conventional techniques described in, e.g., Remington:The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams& Wilkins, Philadelphia, Pa. (2001).

The disclosure further provides methods of treating any disease ordisorder in which expression, improper expression (e.g., overexpression)or increased activity of a PD-1 protein causes or contributes to thepathological effects of the disease, or a decrease in PD-1 proteinlevels or activity has a therapeutic benefit in mammals, such as humans.The disclosure also provides a method of treating a cancer or aninfectious disease in a mammal. Mammals include, e.g., mice, rats,rabbits, dogs, cats, cows, horses, non-human primates, and humans. Themethod comprises administering the aforementioned composition to amammal having a cancer or an infectious disease, whereupon the cancer orinfectious disease is treated in the mammal. As discussed herein, PD-1is abnormally expressed in a variety of cancers (see, e.g., Brown et al,J. Immunol., 170: 1257-1266 (2003); and Flies et. al, Yale Journal ofBiology and Medicine, 84: 409-421 (2011)), and PD-L1 expression in somerenal cell carcinoma patients correlates with tumor aggressiveness.

The disclosure further provides methods of enhancing an immune responseor increasing the activity of an immune cell in a mammal having adisorder that is responsive to PD-1 inhibition. In some embodiments,such methods include administering an effective amount of any PD-1binding agent or antibody agent described herein. In some embodiments,administration of a PD-1 binding agent enhances or increases an immuneresponse or immune cell activity in a mammal or tissue thereof. In someembodiments, an immune response is a humoral or cell mediated immuneresponse. In some embodiments, an immune response is a CD4 or CD8 T cellresponse. In some embodiments, an immune response is a B cell response.

Inventive methods and compositions described herein can be used to treatany type of cancer known in the art, such as, for example, melanoma,renal cell carcinoma, lung cancer, bladder cancer, breast cancer,cervical cancer, colon cancer, gall bladder cancer, laryngeal cancer,liver cancer, thyroid cancer, stomach cancer, salivary gland cancer,prostate cancer, pancreatic cancer, adenocarcinoma (e.g., adenocarcinomaof the lung), or Merkel cell carcinoma (see, e.g., Bhatia et al., Curr.Oncol. Rep., 13(6): 488-497 (2011)). In some embodiments, a cancer isendometrial cancer, breast cancer, ovarian cancer, cervical cancer,fallopian tube cancer, testicular cancer, primary peritoneal cancer,colon cancer, colorectal cancer, stomach cancer, small intestine cancer,squamous cell carcinoma of the anogenital region, melanoma, renal cellcarcinoma, lung cancer, non-small cell lung cancer, squamous cellcarcinoma of the lung, stomach cancer, bladder cancer, gall bladdercancer, liver cancer, thyroid cancer, laryngeal cancer, salivary glandcancer, esophageal cancer, head and neck cancer, squamous cell carcinomaof the head and neck, adenocarcinoma, adenocarcinoma of the lung,prostate cancer, pancreatic cancer, mesothelioma, Merkel cell carcinoma,sarcoma, glioblastoma, or hematological cancer (e.g., multiple myeloma,B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma/primary mediastinalB-cell lymphoma, or chronic myelogenous leukemia). In some embodiments,a cancer to be treated with the inventive methods and/or compositionsdescribed herein is characterized by microsatellite instability or lackthereof. Microsatellite instability (“MSI”) is or comprises a changethat in the DNA of certain cells (such as tumor cells) in which thenumber of repeats of microsatellites (short, repeated sequences of DNA)is different than the number of repeats that was contained in the DNAfrom which it was inherited. Microsatellite instability arises from afailure to repair replication-associated errors due to a defective DNAmismatch repair (MMR) system. This failure allows persistence ofmismatch mutations all over the genome, but especially in regions ofrepetitive DNA known as microsatellites, leading to increased mutationalload. It has been demonstrated that at least some tumors characterizedby MSI-H have improved responses to certain anti-PD-1 agents (Le et al.,(2015) N. Engl. J. Med. 372(26):2509-2520; Westdorp et al., (2016)Cancer Immunol. Immunother. 65(10):1249-1259).

In some embodiments, a cancer has a microsatellite instability status ofhigh microsatellite instability (e.g., MSI-H status). In someembodiments, a cancer has a microsatellite instability status of lowmicrosatellite instability (e.g., MSI-L status). In some embodiments, acancer has a microsatellite instability status of microsatellite stable(e.g., MSS status). In some embodiments microsatellite instabilitystatus is assessed by a next generation sequencing (NGS)-based assay, animmunohistochemistry (IHC)-based assay, and/or a PCR-based assay. Insome embodiments, microsatellite instability is detected by NGS. In someembodiments, microsatellite instability is detected by IHC. In someembodiments, microsatellite instability is detected by PCR.

In embodiments, the cancer is associated with a high tumor mutationburden (TMB). In some embodiments, the cancer is associated with highTMB and MSI-H. In some embodiments, the cancer is associated with highTMB and MSI-L or MSS. In some embodiments, the cancer is endometrialcancer associated with high TMB. In some related embodiments, theendometrial cancer is associated with high TMB and MSI-H. In somerelated embodiments, the endometrial cancer is associated with high TMBand MSI-L or MSS.

In some embodiments, a cancer is a mismatch repair deficient cancer.Microsatellite instability may arise from a failure to repairreplication-associated errors due to a defective DNA mismatch repair(MMR) system. This failure allows persistence of mismatch mutations allover the genome, but especially in regions of repetitive DNA known asmicrosatellites, leading to increased mutational load that may improveresponses to certain anti-PD-1 agents. Id. In some embodiments, a canceris a hypermutated cancer. In some embodiments, a cancer harbors amutation in polymerase epsilon (POLE).

The inventive methods can be used to treat any type of infectiousdisease (i.e., a disease or disorder caused by a bacterium, a virus, afungus, or a parasite). Examples of infectious diseases that can betreated by the inventive method include, but are not limited to,diseases caused by a human immunodeficiency virus (HIV), a respiratorysyncytial virus (RSV), an influenza virus, a dengue virus, a hepatitis Bvirus (HBV, or a hepatitis C virus (HCV)).

The inventive methods can be used to treat any type of autoimmunedisease (i.e., as disease or disorder caused by immune systemover-activity in which the body attacks and damages its own tissues),such as those described in, for example, MacKay I. R. and Rose N. R.,eds., The Autoimmune Diseases, Fifth Edition, Academic Press, Waltham,Mass. (2014). Examples of autoimmune diseases that can be treated by theinventive method include, but are not limited to, multiple sclerosis,type 1 diabetes mellitus, rheumatoid arthritis, scleroderma, Crohn'sdisease, psoriasis, systemic lupus erythematosus (SLE), and ulcerativecolitis.

Administration of a composition comprising an inventive immunoglobulinheavy chain polypeptide, an inventive immunoglobulin light chainpolypeptide, an inventive PD-1-binding agent, an inventive nucleic acidsequence encoding any of the foregoing, or an inventive vectorcomprising an inventive nucleic acid sequence induces an immune responseagainst a cancer or infectious disease in a mammal. Mammals include,e.g., mice, rats, rabbits, dogs, cats, cows, horses, non-human primates,and humans. An “immune response” can entail, for example, antibodyproduction and/or the activation of immune effector cells (e.g.,T-cells).

As used herein, the terms “treatment,” “treating,” and the like refer toobtaining a desired pharmacologic and/or physiologic effect. In someembodiments, the effect is therapeutic, i.e., the effect partially orcompletely cures a disease and/or adverse symptom attributable to thedisease. To this end, the inventive method comprises administering a“therapeutically effective amount” of the PD-1-binding agent. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve a desiredtherapeutic result. The therapeutically effective amount may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the PD-1-binding agent to elicit adesired response in the individual. For example, a therapeuticallyeffective amount of a PD-1-binding agent is an amount which decreasesPD-1 protein bioactivity in a human and/or enhances the immune responseagainst a cancer or infectious disease.

Additionally or alternatively, the pharmacologic and/or physiologiceffect may be prophylactic, i.e., the effect completely or partiallyprevents a disease or symptom thereof. In this respect, the inventivemethod comprises administering a “prophylactically effective amount” ofan anti-PD-1 antibody agent. A “prophylactically effective amount”refers to an amount effective, at dosages and for periods of timenecessary, to achieve a desired prophylactic result (e.g., prevention ofdisease onset).

A typical dose can be, for example, in the range of 1 μg/kg to 20 mg/kgof animal or human body weight; however, doses below or above thisexemplary range are within the scope of the invention. The dailyparenteral dose can be about 0.00001 μg/kg to about 20 mg/kg of totalbody weight (e.g., about 0.001 μg/kg, about 0.1 μg/kg, about 1 μg/kg,about 5 μg/kg, about 10 μg/kg, about 100 μg/kg, about 500 μg/kg, about 1mg/kg, about 5 mg/kg, about 10 mg/kg, or a range defined by any two ofthe foregoing values). In some embodiments, from about 0.1 μg/kg toabout 10 mg/kg of total body weight (e.g., about 0.5 μg/kg, about 1μg/kg, about 50 μg/kg, about 150 μg/kg, about 300 μg/kg, about 750μg/kg, about 1.5 mg/kg, about 5 mg/kg, or a range defined by any two ofthe foregoing values). In some embodiments, from about 1 μg/kg to 5mg/kg of total body weight (e.g., about 3 μg/kg, about 15 μg/kg, about75 μg/kg, about 300 μg/kg, about 900 μg/kg, about 2 mg/kg, about 4mg/kg, or a range defined by any two of the foregoing values). In someembodiments, from about 0.5 to 15 mg/kg body weight per day (e.g., about1 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 6 mg/kg, about 9 mg/kg,about 11 mg/kg, about 13 mg/kg, or a range defined by any two of theforegoing values). Therapeutic or prophylactic efficacy can be monitoredby periodic assessment of treated patients. For repeated administrationsover several days or longer, depending on the condition, the treatmentcan be repeated until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful and are within the scope ofthe invention. The desired dosage can be delivered by a single bolusadministration of the composition, by multiple bolus administrations ofthe composition, or by continuous infusion administration of thecomposition.

Composition(s) comprising an effective amount of an inventiveimmunoglobulin heavy chain polypeptide, an inventive immunoglobulinlight chain polypeptide, an inventive PD-1-binding agent, an inventivenucleic acid sequence encoding any of the foregoing, or an inventivevector comprising an inventive nucleic acid sequence can be administeredto a mammal using standard administration techniques, including oral,ocular, parenteral, intravenous, intraperitoneal, subcutaneous,pulmonary, bronchial, buccal, intradermal, interdermal, transdermal,topical, intramuscular, intranasal, buccal, sublingual, enteral,intra-arterial, intragastric, within a specific organ (e.g.,intrahepatic), rectally, subcutaneously, sublingual, tracheal, vaginal,vitreal, intramedullar, intrathecal, intraventricular, mucosal orsuppository administration. In some embodiments, a composition issuitable for parenteral administration. The term “parenteral,” as usedherein, includes intravenous, intramuscular, subcutaneous, rectal,vaginal, and intraperitoneal administration. In some embodiments, thecomposition is administered to a mammal using peripheral systemicdelivery by intravenous, intraperitoneal, or subcutaneous injection.Mammals include, e.g., mice, rats, rabbits, dogs, cats, cows, horses,non-human primates, and humans.

Once administered to a mammal (e.g., a human), the biological activityof an anti-PD-1 antibody agent can be measured by any suitable methodknown in the art. For example, the biological activity can be assessedby determining the stability of a particular PD-1-binding agent. In someembodiments, an anti-PD-1 antibody agent has an in vivo half-lifebetween about 30 minutes and 45 days (e.g., about 30 minutes, about 45minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours,about 10 hours, about 12 hours, about 1 day, about 5 days, about 10days, about 15 days, about 25 days, about 35 days, about 40 days, about45 days, or a range defined by any two of the foregoing values). In someembodiments, an anti-PD-1 antibody agent has an in vivo half lifebetween about 2 hours and 20 days (e.g., about 5 hours, about 10 hours,about 15 hours, about 20 hours, about 2 days, about 3 days, about 7days, about 12 days, about 14 days, about 17 days, about 19 days, or arange defined by any two of the foregoing values). In some embodiments,the PD-1-binding agent has an in vivo half life between about 10 daysand about 40 days (e.g., about 10 days, about 13 days, about 16 days,about 18 days, about 20 days, about 23 days, about 26 days, about 29days, about 30 days, about 33 days, about 37 days, about 38 days, about39 days, about 40 days, or a range defined by any two of the foregoingvalues).

The stability of an anti-PD-1 antibody agent can be measured using anyother suitable assay known in the art, such as, for example, measuringserum half-life, differential scanning calorimetry (DSC), thermal shiftassays, and pulse-chase assays. Other methods of measuring proteinstability in vivo and in vitro that can be used in the context of theinvention are described in, for example, Protein Stability and Folding,B. A. Shirley (ed.), Human Press, Totowa, N.J. (1995); ProteinStructure, Stability, and Interactions (Methods in Molecular Biology),Shiver J. W. (ed.), Humana Press, New York, N.Y. (2010); and Ignatova,Microb. Cell Fact., 4: 23 (2005).

The stability of an anti-PD-1 antibody agent can be measured in terms ofthe transition mid-point value (T_(m)), which is the temperature where50% of the amino acid sequence is in its native confirmation, and theother 50% is denatured. In general, the higher the T_(m), the morestable the protein. In some embodiments, an inventive PD-1 binding agentcomprises a transition mid-point value (T_(m)) in vitro of about 60-100°C. For example, an anti-PD-1 antibody agent can comprise a T_(m) invitro of about 65-80° C. (e.g., 66° C., 68° C., 70° C., 71° C., 75° C.,or 79° C.), about 80-90° C. (e.g., about 81° C., 85° C., or 89° C.), orabout 90-100° C. (e.g., about 91° C., about 95° C., or about 99° C.).

The biological activity of a particular anti-PD-1 antibody agent alsocan be assessed by determining its binding affinity to a PD-1 protein oran epitope thereof. The term “affinity” refers to the equilibriumconstant for the reversible binding of two agents and is expressed asthe dissociation constant (K_(D)). Affinity of a binding agent to aligand, such as affinity of an antibody for an epitope, can be, forexample, from about 1 picomolar (pM) to about 100 micromolar (μM) (e.g.,from about 1 picomolar (pM) to about 1 nanomolar (nM), from about 1 nMto about 1 micromolar (μM), or from about 1 μM to about 100 μM). In someembodiments, the PD-1-binding agent can bind to an PD-1 protein with aK_(D) less than or equal to 1 nanomolar (e.g., 0.9 nM, 0.8 nM, 0.7 nM,0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.05 nM, 0.025 nM, 0.01nM, 0.001 nM, or a range defined by any two of the foregoing values). Insome embodiments, the PD-1-binding agent can bind to PD-1 with a K_(D)less than or equal to 200 pM (e.g., 190 pM, 175 pM, 150 pM, 125 pM, 110pM, 100 pM, 90 pM, 80 pM, 75 pM, 60 pM, 50 pM, 40 pM, 30 pM, 25 pM, 20pM, 15 pM, 10 pM, 5 pM, 1 pM, or a range defined by any two of theforegoing values). Immunoglobulin affinity for an antigen or epitope ofinterest can be measured using any art-recognized assay. Such methodsinclude, for example, fluorescence activated cell sorting (FACS),separable beads (e.g., magnetic beads), surface plasmon resonance (SPR),solution phase competition (KINEXA™), antigen panning, and/or ELISA(see, e.g., Janeway et al. (eds.), Immunobiology, 5th ed., GarlandPublishing, New York, N.Y., 2001).

An anti-PD-1 antibody agent may be administered alone or in combinationwith other drugs (e.g., as an adjuvant). For example, a PD-1-bindingagent can be administered in combination with other agents for thetreatment or prevention of the diseases disclosed herein, such as agentsthat are cytotoxic to cancer cells, modulate the immunogenicity ofcancer cells, or promote immune responses to cancer cells. In thisrespect, for example, an anti-PD-1 antibody agent can be used incombination with at least one other anticancer agent including, forexample, any chemotherapeutic agent known in the art, ionizationradiation, small molecule anticancer agents, cancer vaccines, biologicaltherapies (e.g., other monoclonal antibodies, cancer-killing viruses,gene therapy, and adoptive T-cell transfer), and/or surgery. In someembodiments, a subject (e.g., a mammal, e.g., a human) for treatmentwith an anti-PD-1 antibody agent has been treated or will be treatedwith chemotherapy (e.g., platinum-based chemotherapy). In someembodiments, a chemotherapeutic agent is actinomycin, all-trans retinoicacid, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin,capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine,daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin,epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea,idarubicin, imatinib, irinotecan, mechlorethamine, mercaptopurine,methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed,teniposide, tioguanine, topotecan, valrubicin, vemurafenib, vinblastine,vincristine, vindesine, or vinorelbine. In some such embodiments, achemotherapeutic agent is a platinum-based chemotherapeutic agent, suchas cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatintetranitrate, phenanthriplatin, picoplatin, or satraplatin. In some suchembodiments, a chemotherapeutic agent is a folate antimetabolite such aspemetrexed. In some embodiments, a subject (e.g., a mammal, e.g. ahuman) for treatment with an anti-PD-1 antibody agent has been treatedor will be treated with an anti-angiogenic agent, for example,bevacizumab, itraconazole, carboxyamidotriazole, TNP-470, fumagillin,CM101, IL-12, platelet factor-4, suramin, SU5416, thrombospondin,angiostatic steroids, heparin, cartilage-derived angiogenesis inhibitoryfactor (e.g. peptide troponin I and chondromodulin I), matrixmetalloproteinase inhibitor, angiostatin, endostatin,2-methoxyestradiol, tecogalan, tetrathiomolybdate, thrombospondin,thalidomide, prolactin, αVβ3 inhibitor, lenalidomide, linomide,ramucirumab, tasquinimod, ranibizumab, sorafenib, sunitinib, pazopanib,everolimus, tissue inhibitors of metalloproteases (TIMP1 and TIMP2),bFGF soluble receptor, transforming growth factor beta, interferonalpha, interferon beta, soluble KDR and FLT-1 receptors, placentalproliferin-related protein, pazopanib, sunitinib, sorafenib, axitinib,ponatinib, cabozantinib, regorafenib, vandetanib, lenvatinib, semaxanib,SU6668, vatalanib, tivozanib, cediranib, protamine, heparin, steroids,ascorbic acid ethers, sulfated polysaccharide DS 4152, fumagillin, AGM12470, neovastat, R04929097, MRK-003, MK-0752, PF03084014, MEDI0639,curcumin, 3,3′-diindolylmethane (DIM), resveratrol,3,5-bis(2,4-difluorobenzylidene)-4-piperidone (DiFiD) andepigallocatechin-3-gallate (EGCG), honokiol, Flt2-11, CBO-P11, Je-11,V1, and any combination thereof. In some embodiments, an anti-PD-1antibody agent can be used in combination with an anti-inflammatoryagent including, for example, corticosteroids (e.g., prednisone andfluticasone) and non-steroidal anti-inflammatory drugs (NSAIDs) (e.g.,aspirin, ibuprofen, and naproxen).

In some embodiments, an anti-PD-1 antibody agent is used to treat aninfectious disease. When the inventive method treats an infectiousdisease, an anti-PD-1 antibody agent can be administered in combinationwith at least one antibacterial agent or at least one anti-viral agent.In this respect, the anti-bacterial agent can be any suitable antibioticknown in the art. The anti-viral agent can be any vaccine of anysuitable type that specifically targets a particular virus (e.g.,live-attenuated vaccines, subunit vaccines, recombinant vector vaccines,and small molecule anti-viral therapies (e.g., viral replicationinhibitors and nucleoside analogs).

In some embodiments, an anti-PD-1 antibody agent is used to treat anautoimmune disease. In some embodiments, an autoimmune disease ismultiple sclerosis, type 1 diabetes mellitus, rheumatoid arthritis,scleroderma, Crohn's disease, psoriasis, systemic lupus erythematosus(SLE), or ulcerative colitis. When the inventive method treats anautoimmune disease, an anti-PD-1 antibody agent can be used incombination with an anti-inflammatory agent including, for example,corticosteroids (e.g., prednisone and fluticasone) and non-steroidalanti-inflammatory drugs (NSAIDs) (e.g., aspirin, ibuprofen, andnaproxen).

In some embodiments, an anti-PD-1 antibody agent can be administered incombination with other agents that inhibit immune checkpoint pathways.For example, an inventive PD-1 binding agent can be administered incombination with agents that inhibit or antagonize the CTLA-4, TIM-3 orLAG-3 pathways. Combination treatments that simultaneously target two ormore of these immune checkpoint pathways have demonstrated improved andpotentially synergistic antitumor activity (see, e.g., Sakuishi et al,J. Exp. Med., 207: 2187-2194 (2010); Ngiow et al, Cancer Res., 71:3540-3551 (2011); and Woo et al., Cancer Res., 72: 917-927 (2012)). Insome embodiments, an inventive PD-1 binding agent is administered incombination with an antibody that binds to TIM-3 and/or an antibody thatbinds to LAG-3. In this respect, the inventive method of treating acancer or an infectious disease in a mammal can further compriseadministering to the mammal a composition comprising (i) an antibodythat binds to a TIM-3 protein and (ii) a pharmaceutically acceptablecarrier or a composition comprising (i) an antibody that binds to aLAG-3 protein and (ii) a pharmaceutically acceptable carrier. Exemplaryantibody agents specific for LAG-3 and TIM-3 are described inWO2016/126858 and WO2016/161270, respectively, both of which are herebyincorporated by reference. In some embodiments, an anti-TIM-3 antibodyagent can be used in combination with an anti-inflammatory agentincluding, for example, corticosteroids (e.g., prednisone andfluticasone) and non-steroidal anti-inflammatory drugs (NSAIDs) (e.g.,aspirin, ibuprofen, and naproxen).

In some embodiments, an anti-PD-1 antibody agent is administered incombination with an agent that inhibits LAG-3 signaling and/or an agentthat inhibits TIM-3 signaling. In some embodiments, an anti-PD-1antibody agent is administered to a subject that has been administeredor will be administered an agent that inhibits LAG-3 signaling, suchthat the subject receives treatment with both. In some embodiments, ananti-PD-1 antibody agent is administered to a subject that has beenadministered or will be administered an agent that inhibits TIM-3signaling, such that the subject receives treatment with both. In someembodiments, a mammal that receives treatment an anti-PD-1 antibodyagent has been or will receive treatment with an agent that inhibitsTIM-3 and an agent that inhibits LAG-3, such that the mammal receivesall three. In some embodiments, an anti-PD-1 antibody agent isadministered in combination with an antibody that binds to LAG-3 and/oran antibody that binds to TIM-3.

In some embodiments, a subject is receiving or will receive one or moreadditional therapies in combination with an anti-PD-1 antibody agent. Insome embodiments, an additional therapy is a PARP inhibitor. In someembodiments, a PARP inhibitor is ABT-767, AZD 2461, BGB-290, BGP 15, CEP8983, CEP 9722, DR 2313, E7016, E7449, fluzoparib (SHR 3162), IMP 4297,INO1001, JPI 289, JPI 547, monoclonal antibody B3-LysPE40 conjugate, MP124, niraparib (ZEJULA) (MK-4827), NU 1025, NU 1064, NU 1076, NU1085,olaparib (AZD2281), ONO2231, PD 128763, R 503, R554, rucaparib (RUBRACA)(AG-014699, PF-01367338), SBP 101, SC 101914, Simmiparib, talazoparib(BMN-673), veliparib (ABT-888), WW 46,2-(4-(Trifluoromethyl)phenyl)-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-4-ol,and salts or derivatives thereof. In some embodiments, a PARP inhibitoris niraparib, olaparib, rucaparib, talazoparib, and veliparib. In someembodiments, additional therapies include treatment with a compositionthat delivers an agent that inhibits TIM-3 or LAG-3 and treatment with aPARP inhibitor such that the subject receives treatment with all three.In some embodiments, additional therapies include treatment with acomposition that delivers an agent that inhibits TIM-3, treatment with acomposition that delivers an agent that inhibits LAG-3, and treatmentwith a PARP inhibitor such that the subject receives treatment with allfour.

In addition to therapeutic uses, an anti-PD-1 antibody agent asdescribed herein can be used in diagnostic or research applications. Inthis respect, an anti-PD-1 antibody agent can be used in a method todiagnose a cancer or infectious disease. In a similar manner, ananti-PD-1 antibody agent can be used in an assay to monitor PD-1 proteinlevels in a subject being tested for a disease or disorder that isassociated with abnormal PD-1 expression. Research applications include,for example, methods that utilize an anti-PD-1 antibody agent and alabel to detect a PD-1 protein in a sample, e.g., in a human body fluidor in a cell or tissue extract. An anti-PD-1 antibody agent can be usedwith or without modification, such as covalent or non-covalent labelingwith a detectable moiety. For example, the detectable moiety can be aradioisotope (e.g., ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I), a fluorescent orchemiluminescent compound (e.g., fluorescein isothiocyanate, rhodamine,or luciferin), an enzyme (e.g., alkaline phosphatase,beta-galactosidase, or horseradish peroxidase), or prosthetic groups.Any method known in the art for separately conjugating anantigen-binding agent (e.g., an antibody) to a detectable moiety may beemployed in the context of the invention (see, e.g., Hunter et al,Nature, 194: 495-496 (1962); David et al, Biochemistry, 13: 1014-1021(1974); Pain et al, J. Immunol. Meth., 40: 219-230 (1981); and Nygren,J. Histochem. and Cytochem., 30: 407-412 (1982)).

PD-1 protein levels can be measured using an anti-PD-1 antibody agent asdescribed herein using any suitable method known in the art. Suchmethods include, for example, radioimmunoassay (RIA), and FACS. Normalor standard expression values of PD-1 protein can be established usingany suitable technique, e.g., by combining a sample comprising, orsuspected of comprising, a PD-1 polypeptide with a PD-1-specificantibody under conditions suitable to form an antigen-antibody complex.The antibody is directly or indirectly labeled with a detectablesubstance to facilitate detection of the bound or unbound antibody.Suitable detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials, and radioactivematerials (see, e.g., Zola, Monoclonal Antibodies: A Manual ofTechniques, CRC Press, Inc. (1987)). The amount of PD-1 polypeptideexpressed in a sample is then compared with a standard value.

An anti-PD-1 antibody agent can be provided in a kit, i.e., a packagedcombination of reagents in predetermined amounts with instructions forperforming a diagnostic assay. If the PD-1-binding agent is labeled withan enzyme, the kit desirably includes substrates and cofactors requiredby the enzyme (e.g., a substrate precursor which provides a detectablechromophore or fluorophore). In addition, other additives may beincluded in the kit, such as stabilizers, buffers (e.g., a blockingbuffer or lysis buffer), and the like. The relative amounts of thevarious reagents can be varied to provide for concentrations in solutionof the reagents which substantially optimize the sensitivity of theassay. The reagents may be provided as dry powders (typicallylyophilized), including excipients which on dissolution will provide areagent solution having the appropriate concentration.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments, which are given forillustration of the invention and are not intended to be limitingthereof.

EXEMPLIFICATION Example 1—Description of Certain Exemplary Anti-PD-1Antibodies

This example describes particular anti-PD-1 antibody heavy chainpolypeptide and light chain polypeptide sequences and nucleic acidsencoding the same.

An anti-PD-1 antibody heavy chain polypeptide (CDR sequences)(SEQ ID NO: 1) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISGGGSYTYYQDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASPYYAMDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDEMPSNTKVDKRVESKYGPPCPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAn anti-PD-1 antibody light chain polypeptide (CDR sequences) (SEQ ID NO: 2) DIQLTQSPSFLSAYVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTLHTGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQHYSSYPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGECAn anti-PD-1 antibody heavy chain polypeptide with a signal sequence (SEQ ID NO: 5) MEFGLSWLFLVAILKGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISGGGSYTYYQDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASPYYAMDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDEEKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGKAn anti-PD-1 antibody light chain polypeptide with a signal sequence (SEQ ID NO: 6) MDMRVPAQLLGLLLLWLPGARCDIQLTQSPSFLSAYVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTLHTGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQHYSSYPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECNucleotide sequence encoding anti-PD-1 antibody heavy chain polypeptide (SEQ ID NO: 3) GAG GTG CAG CTG TTG GAG TCT GGG GGA GGC TTG GTA CAG CCTGGG GGG TCC CTG AGA CTC TCC TGT GCA GCC TCT GGA TTC ACTTTC AGT AGC TAT GAC ATG TCT TGG GTC CGC CAG GCT CCA GGGAAG GGG CTG GAG TGG GTC TCA ACC ATT AGT GGT GGT GGT AGTTAC ACC TAC TAT CAA GAC AGT GTG AAG GGG CGG TTC ACC ATCTCC AGA GAC AAT TCC AAG AAC ACG CTG TAT CTG CAA ATG AACAGC CTG AGA GCC GAG GAC ACG GCC GTA TAT TAC TGT GCG TCCCCT TAC TAT GCT ATG GAC TAC TGG GGG CAA GGG ACC ACG GTCACC GTC TCC TCA GCA TCC ACC AAG GGC CCA TCG GTC TTC CCGCTA GCA CCC TGC TCC AGG AGC ACC TCC GAG AGC ACA GCC GCCCTG GGC TGC CTG GTC AAG GAC TAC TTC CCC GAA CCA GTG ACGGTG TCG TGG AAC TCA GGC GCC CTG ACC AGC GGC GTG CAC ACCTTC CCG GCT GTC CTA CAG TCC TCA GGA CTC TAC TCC CTC AGC AGCGTG GTG ACC GTG CCC TCC AGC AGC TTG GGC ACG AAG ACC TACACC TGC AAC GTA GAT CAC AAG CCC AGC AAC ACC AAG GTG GACAAG AGA GTT GAG TCC AAA TAT GGT CCC CCA TGC CCA CCA TGCCCA GCA CCT GAG TTC CTG GGG GGA CCA TCA GTC TTC CTG TTCCCC CCA AAA CCC AAG GAC ACT CTC ATG ATC TCC CGG ACC CCTGAG GTC ACG TGC GTG GTG GTG GAC GTG AGC CAG GAA GAC CCCGAG GTC CAG TTC AAC TGG TAC GTG GAT GGC GTG GAG GTG CATAAT GCC AAG ACA AAG CCG CGG GAG GAG CAG TTC AAC AGC ACGTAC CGT GTG GTC AGC GTC CTC ACC GTC CTG CAC CAG GAC TGGCTG AAC GGC AAG GAG TAC AAG TGC AAG GTC TCC AAC AAA GGCCTC CCG TCC TCC ATC GAG AAA ACC ATC TCC AAA GCC AAA GGGCAG CCC CGA GAG CCA CAG GTG TAC ACC CTG CCC CCA TCC CAGGAG GAG ATG ACC AAG AAC CAG GTC AGC CTG ACC TGC CTG GTCAAA GGC TTC TAC CCC AGC GAC ATC GCC GTG GAG TGG GAG AGCAAT GGG CAG CCG GAG AAC AAC TAC AAG ACC ACG CCT CCC GTGCTG GAC TCC GAC GGC TCC TTC TTC CTC TAC AGC AGG CTA ACCGTG GAC AAG AGC AGG TGG CAG GAG GGG AAT GTC TTC TCA TGCTCC GTG ATG CAT GAG GCT CTG CAC AAC CAC TAC ACA CAG AAGAGC CTC TCC CTG TCT CTG GGT AAANucleotide sequence encoding an anti-PD-1 antibody light chain polypeptide (SEQ ID NO: 4)GAC ATC CAG TTG ACC CAG TCT CCA TCC TTC CTG TCT GCA TAT GTAGGA GAC AGA GTC ACC ATC ACT TGC AAG GCC AGT CAG GAT GTGGGT ACT GCT GTA GCC TGG TAT CAG CAA AAA CCA GGG AAA GCCCCT AAG CTC CTG ATC TAT TGG GCA TCC ACC CTG CAC ACT GGGGTC CCA TCA AGG TTC AGC GGC AGT GGA TCT GGG ACA GAA TTCACT CTC ACA ATC AGC AGC CTG CAG CCT GAA GAT TTT GCA ACTTAT TAC TGT CAG CAT TAT AGC AGC TAT CCG TGG ACG TTT GGCCAG GGG ACC AAG CTG GAG ATC AAA CGG ACT GTG GCT GCA CCATCT GTC TTC ATC TTC CCG CCA TCT GAT GAG CAA TTG AAA TCT GGAACT GCC TCT GTT GTG TGC CTG CTG AAT AAC TTC TAT CCC AGA GAGGCC AAA GTA CAG TGG AAG GTG GAT AAC GCC CTC CAA TCG GGTAAC TCC CAG GAG AGT GTC ACA GAG CAG GAC AGC AAG GAC AGCACC TAC AGC CTC AGC AGC ACC CTG ACG CTG AGC AAA GCA GACTAC GAG AAA CAC AAA GTC TAC GCC TGC GAA GTC ACC CAT CAGGGC CTC AGC TCG CCC GTC ACA AAG AGC TTC AAC AGG GGA GAG TGT

The sequences above describe an exemplary humanized monoclonal anti-PD-1antibody utilizing a human IGHG4*01 heavy chain gene, and a humanIGKC*01 kappa light chain gene, as scaffolds. There is a single Ser toPro point mutation in the hinge region of the IgG4 heavy chain. Thismutation is at the canonical S228 position, corresponding to residue 243in SEQ ID NO: 5, which includes the signal sequence. Without wishing tobe bound by theory, it is envisioned that this point mutation serves tostabilize the hinge of the antibody heavy chain.

Biophysical and biochemical characterization of this exemplary humanizedmonoclonal anti-PD-1 antibody is consistent with the expected disulfidelinkage pattern for an IgG4 molecule. The residues involved in theexpected inter- and intrachain disulfide linkages are tabulated below(Tables 1 and 2).

TABLE 1 Expected residues involved in disulfide linkages of an exemplaryanti-PD-1 antibody agent heavy chain having an amino acid sequence asset forth in SEQ ID NO: 1. Cysteine residue ID anti-PD-1 mAb HC Residueafter Edelman^(a) (position in SEQ ID NO: 1) I 22 II 96 III 130 IV 143 V199 VI 222 VII 225 VIII 257 IX 317 X 363 XI 421

TABLE 2 Expected residues involved in disulfide linkages of an exemplaryanti-PD-1 antibody agent light chain having an amino acid sequence asset forth in SEQ ID NO: 2. Cysteine residue ID anti-PD-1 mAb LC Residueafter Edelman^(a) (position in SEQ ID NO: 2) I 23 II 88 III 134 IV 194 V214

This exemplary anti-PD-1 antibody exhibits an occupied N-glycosylationsite at asparagine residue 293 in the CH2 domain of each heavy chain inthe mature protein sequence (SEQ ID NO:1). The expressed N-glycosylationat this site is a mixture of oligosaccharide species typically observedon IgGs expressed in mammalian cell culture, for example, shown below isthe relative abundance of glycan species from a preparation of thisexemplary anti-PD-1 antibody cultured in Chinese Hamster Ovary (CHO)cells (Table 3).

TABLE 3 Glycan Analysis of an anti-PD-1 antibody binding agent Abundance(% of total Species oligosaccharide) Description of Glycan G0 <0.1%Nonfucosylated agalactobiantennary complex-type oligosaccharide G0F19.5% Core fucosylated agalactobiantennary complex type oligosaccharideG1  0.1% Nonfucosylated monogalactosylated biantennary complex typeoligosaccharide G1F 45.6% Core fucosylated monogalactosylatedbiantennary complex type oligosaccharide G2F 27.4% Core fucosylatedgalactosylated biantennary complex type oligosaccharide M5  0.5%Oligomannosidic N-glycan, Man₅GlcNAc₂

Example 2—Binding of an Exemplary Anti-PD-1 Antibody to Recombinant PD-1

This example describes binding of an exemplary anti-PD-1 antibody(having heavy and light chains as set forth in SEQ ID NOs: 1 & 2,respectively) to recombinant PD-1 polypeptides. Specifically, thisexample demonstrates high affinity binding of an exemplary antibody tosoluble PD-1 fusions and cell-expressed recombinant PD-1 as determinedusing surface plasmon resonance (SPR) and flow cytometry, respectively.

SPR analyses were carried out using a Biacore T200 system and kineticconstants were determined using Biacore T200 Evaluation software.Experimental parameters were chosen such that saturation was reached atthe highest antigen concentrations and R_(max) values were kept under100 response units (RU). GE anti-human IgG (Fc-specific) was immobilizedon a Biacore CM5 chip. An exemplary anti-PD-1 antibody (having heavy andlight chains as set forth in SEQ ID NOs: 1 & 2, respectively) was thencaptured onto this surface using EDC-activated amine coupling chemistry.Next, dimeric human or cynomolgus monkey PD-1 fusion proteins (fusedwith mouse IgG2a Fc) in a two-fold serial dilution series were flowedover the captured exemplary antibody and dissociation was monitored.Capture and analyte binding were performed in HBS-EP+ buffer. Chips wereregenerated between each run using 3 M MgCl2. The resulting sensorgramswere fitted globally using a 1:1 binding model to calculate on- andoff-rates (k_(assoc) and k_(dissoc), respectively) and dissociationconstants as a measure of overall affinity (K_(D)). SPR measurementsdemonstrated that an exemplary anti-PD-1 antibody binds human andcynomolgus PD-1 with a fast association rate, a slow dissociation rate,and a high overall affinity (Table 4). Moreover, binding kinetics tohuman and cynomolgus monkey PD-1 were similar, with less than a 2-folddifference in K_(D) values.

Flow cytometry studies were performed with CHO-K1 cell line clones inwhich either full length native human or cynomolgus monkey PD-1 wasstably transfected. An exemplary anti-PD-1 antibody (having heavy andlight chains as set forth in SEQ ID NOs: 1 & 2, respectively) wasdiluted in 3-fold dilutions. Dilutions of exemplary antibody were addedto human or cynomolgus monkey PD-1 expressing CHO-K1 cells (1E5 cells)and incubated on ice. Cells were washed twice and incubated on ice withPE-conjugated mouse anti-human IgG4 to detect antibody binding. Cellswere washed and resuspended in the presence of propidium iodide toexclude dead cells and fixed and analyzed for fluorescence on a BDFACSArray instrument (BD Biosciences). Data were analyzed for medianfluorescence intensity, graphed, and curves fitted for EC₅₀ valuecalculation in GraphPad Prism (GraphPad Software, Inc.) using anon-linear (sigmoidal) regression analysis. This exemplary anti-PD-1antibody was found to bind to cell-surface human and cynomolgus monkeyPD-1 with an EC₅₀ of 2.0 and 3.4 nM, respectively (Table 4).

TABLE 4 Binding of exemplary anti-PD-1 antibody to PD-1 by SurfacePlasma Resonance (SPR) and PD-1 expressing CHO-K1 cells PD-1 expressingKinetic Parameters (SPR) CHO-K1 cells Species K_(assoc) (Ms)⁻¹K_(dissoc) (s⁻¹) K_(D) (nM) EC₅₀(nM) Human 5.7 × 10⁵ 1.7 × 10⁻⁴ 0.30 2.0Cynomolgus 4.3 × 10⁵ 2.3 × 10⁻⁴ 0.53 3.4 monkey K_(assoc) = associationrate constant; K_(dissoc) = dissociation rate constant; K_(D) =dissociation constant.

This example demonstrates that anti-PD-1 antibodies within the scope ofthe present invention can bind to PD-1 polypeptides with high affinity.

Example 3—Receptor Occupancy of an Exemplary Anti PD-1 Antibody

This example describes the ability of an exemplary anti-PD-1 antibody(having heavy and light chains as set forth in SEQ ID NOs: 1 & 2,respectively) to occupy the native PD-1 expressed on human andcynomolgus monkey peripheral blood mononuclear cells (PBMCs).

For these studies, PBMCs from healthy human donors or cynomolgus monkeyswere used. Human PBMCs were isolated from buffy coats obtained from theIndiana Blood Center and cynomolgus monkey PBMCs were isolated fromperipheral blood collected aseptically into sodium heparin obtained fromWorldwide Primates, Inc. In both cases, separation was by Ficoll usingFicoll-Paque 1.077 and cryopreserved for later use.

On the day of the experiments, the cryopreserved cells were thawed andthe cell concentration was adjusted to 2E6 cells/ml prior to restingovernight at 37° C. in a humidified CO₂ incubator. Cells were thenpelleted and re-suspended in 1 ml of media and recounted. The cellconcentration was adjusted to 4E5 cells/150 μl of media. Human AB Serum(40 μl/ml) was added to block Fc receptors. After centrifugation, thecells were incubated with exemplary anti-PD-1 antibody at 4° C. PBMCswere then washed three times before being split into two conditions. Oneset of cells was incubated with exemplary anti-PD-1 antibody and theother set with IgG4 for 30 minutes at 4° C. The PBMCs were then washedfour times prior to being stained with FITC-labeled anti-CD3 andPE-labeled anti-IgG4 antibodies. The cells were washed and fixed beforeanalysis by flow cytometry. The number of CD3+/IgG4+ cells wasdetermined for each parameter and the percentage of occupancy for theexemplary anti-PD-1 antibody was determined as follows:

-   -   [Number of CD3+/IgG4+ cells for IgG4 treated cells at a given        pre-incubation concentration of anti-PD-1 antibody] divided by    -   [Number of CD3+/IgG4+ cells for exemplary anti-PD-1 antibody        treated cells at a given pre-incubation concentration of        anti-PD-1 antibody]

For PBMCs pre-incubated with exemplary anti-PD-1 antibody, both the IgG4treated as well as anti-PD-1 antibody treated cells generated a highnumber of CD3+/IgG4+ cells. As the level of exemplary anti-PD-1 antibodywas reduced during the pre-incubation step, the number of CD3+/IgG4+cells detected with the IgG4 treated cells steadily decreased,indicative of concentration dependent occupancy of PD-1 by exemplaryanti-PD-1 antibody (FIG. 1).

This example demonstrates that anti-PD-1 antibodies within the scope ofthe present invention can bind natively expressed PD-1 and further thatthe occupancy of PD-1 on PBMCs by exemplary anti-PD-1 antibody isconcentration dependent.

Example 4—an Exemplary Anti-PD-1 Antibody Blocks Interaction BetweenPD-1 and PD-L1 and PD-L2

This example describes the ability of an exemplary anti-PD-1 antibody(having heavy and light chains as set forth in SEQ ID NOs: 1 & 2,respectively) to prevent the interaction between PD-1 and its cognateligands, PD-L1 and PD-L2.

In these studies, human PD-L1 and PD-L2 mouse IgG1 Fc fusion proteinswere expressed, purified, and labeled with DyL650. The dose-response ofbinding of both PD-L1 and PD-L2 to PD-1 CHO-K1 cells was determined. Toquantify blocking of ligand binding to PD-1 CHO-K1 cells, exemplaryanti-PD-1 antibody or IgG4 control antibody in a 3-fold dilution serieswas pre-mixed with PD-L1-mFc-DyL650 or PD-L2-mFc-DyL650. The mixture wasadded to human PD-1 CHO-K1 cells (3E5 cells) and incubated at 4° C.Cells were washed once and re-suspended in the presence of propidiumiodide and DyL650-PD-L1 or DyL650-PD-L2. Binding was analyzed on a BDFACSArray (BD Bioscience), excluding dead cells. Data were analyzed formedian fluorescence intensity and curves were fitted for IC₅₀calculation using non-linear regression analysis in GraphPad Prism(Graphpad Software, Inc.) It was found that, unlike an IgG4 controlantibody, an exemplary anti-PD-1 antibody (having heavy and light chainsas set forth in SEQ ID NOs: 1 & 2, respectively) was able to potentlyinhibit the interaction between PD-1 and both PD-L1 and PD-L2 (Table 5).

TABLE 5 Potency of an exemplary anti-PD-1 antibody to inhibit theinteraction between cell-expressed PD-1 and soluble PD-Ll and PD-L2PD-L1/PD-1 CHO-K1 PD-L2/PD-1 CHO-K1 Antibody competition IC₅₀ (nM)competition IC₅₀ (nM) Exemplary anti-PD-1 1.8 1.5 antibody (having heavyand light chains as set forth in SEQ ID NOs: 1 & 2, respectively)

This example demonstrates that anti-PD-1 antibodies within the scope ofthe present invention can block the binding of PD-1 ligands such asPD-L1 and PD-L2.

Having thus described at least several aspects and embodiments of thisinvention, it is to be appreciated that various alterations,modifications, and improvements will readily be apparent to thoseskilled in the art. Such alterations, modifications, and improvementsare intended to be part of this disclosure, and are intended to bewithin the spirit and scope of the invention. Accordingly, the foregoingdescription are by way of example only and the invention is described indetail by the claims that follow.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

What is claimed is:
 1. A method of treating a disorder in a human thatis responsive to PD-1 inhibition, the method comprising administering tothe human an effective amount of an antibody comprising: a heavy chainpolypeptide comprising the amino acid sequence of SEQ ID NO: 1 and alight chain polypeptide comprising the amino acid sequence of SEQ ID NO:2, whereupon the disorder is treated in the human.
 2. The method ofclaim 1, wherein the disorder is cancer.
 3. The method of claim 2,wherein the cancer is: adenocarcinoma, endometrial cancer, breastcancer, ovarian cancer, cervical cancer, fallopian tube cancer,testicular cancer, primary peritoneal cancer, colon cancer, colorectalcancer, stomach cancer, small intestine cancer, squamous cell carcinomaof the anogenital region, melanoma, renal cell carcinoma, lung cancer,non-small cell lung cancer, adenocarcinoma of the lung, squamous cellcarcinoma of the lung, stomach cancer, bladder cancer, gall bladdercancer, liver cancer, thyroid cancer, laryngeal cancer, salivary glandcancer, esophageal cancer, head and neck cancer, squamous cell carcinomaof the head and neck, prostate cancer, pancreatic cancer, mesothelioma,Merkel cell carcinoma, sarcoma, glioblastoma, and a hematologicalcancer, such as multiple myeloma, B-cell lymphoma, T-cell lymphoma,Hodgkin's lymphoma/primary mediastinal B-cell lymphoma, or chronicmyelogenous leukemia.
 4. The method of claim 2, wherein the cancer: ischaracterized by microsatellite instability, is microsatelliteinstability high (MSI-H), has high tumor mutational burden (TMB), has adefective DNA mismatch repair system, has a defect in a DNA mismatchrepair gene, is a hypermutated cancer, or comprises a mutation inpolymerase epsilon (POLE).